This invention relates to the manipulation of plant gene expression and the production of transgenic plants.
Auxin is an essential plant hormone that regulates diverse processes, such as cell division and elongation, root and leaf development, apical dominance, tropism, and reproduction (Davies, P. J., In: Plant hormones, ed., Davies, P. J., pp. 1-12, Kluwer, Dordrecht, Netherlands, 1995). The auxin response is regulated by a complex signaling network, and reflects a balance between auxin and other synergistical or antagonistical signaling pathways in plant cells (Bellincampi et al., Plant Cell 8: 477-487, 1996; Coenen et al., Trends Plant Sci. 2: 351-356, 1997). A primary event of auxin action is the activation of many early response genes. Extensive studies of the early response gene promoters have identified several auxin responsive cis-elements and trans-acting factors (Abel et al., Plant Physiol. 111: 9-17, 1996; Ulmasov et al., Science 276: 1865-1868, 1997). Although genetic approaches have significantly advanced our understanding of auxin action (Walden et al., Trends Plant Sci. 1: 335-339, 1996; Leyser, Curr. Biol. 8: R305-R307, 1998; Guilfoyle, Trends Plant Sci. 3: 205-207, 1998), the molecular mechanisms underlying signal transduction pathways that control auxin responsive transcription remain largely unknown.
In yeast, worms, insects, and mammals, the primary responses to hormone, growth, and stress signals are mediated by a conserved signaling cascade consisting of three protein kinases, the mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). MAPKKK phosphorylates and activates MAPKK that, in turn, phosphorylates and activates MAPK. The activated MAPK can be translocated into the nucleus where it phosphorylates transcription factors that control gene expression (Herskowitz, Cell 80: 187-197, 1995; Kyriakis et al., J. Biol. Chem. 271: 24313-24316, 1996). Although many plant MAPK, MAPKK, and MAPKKK homologues have been identified based on sequence conservation and functional complementation in yeast, their precise physiological functions in plants are largely unknown (Hirt, Trends Biol. Sci. 2: 11-15, 1997). It also remains unclear whether and how these homologues constitute specific MAPK kinase cascades (Mizoguchi et al., Trends Biotech. 15: 15-19, 1997).
Plants are constantly exposed to environmental stimuli that influence their growth and development. Adverse environmental conditions, including heat, salinity, freezing, and drought, greatly compromise plant productivity and reduce crop yield. Genetic approaches have been taken to enhance plant tolerance to stresses through alteration of osmolytes, osmoprotectants, membrane fatty acids, channels, transcription factors, and enzymes that scavenge active oxygen species by transferring or mutating individual stress target genes. A need in the art therefore exists for developing molecular strategies that enable plants to have resistance or tolerance to adverse environmental conditions.
The invention is based on applicants"" discovery that a mitogen-activated protein kinase kinase kinase (MAPKKK) polypeptide, such as NPK1 of tobacco and the ANPs of Arabidopsis, is involved in signaling the activation of stress protective gene transcription, repression of early auxin response gene transcription, and the alteration of seed development. Accordingly, the invention involves methods of genetically engineering plants to produce altered, agronomic, physiological, or developmental changes in plants by expressing a transgene including DNA encoding a kinase domain of a MAPKKK within the tissues of the plants. In particular, it has been found that it is possible to engineer plants that express a recombinant MAPKKK that are resistant to a broad spectrum of stresses (e.g., drought, increased salinity, heat shock, and freezing temperature), that have repressed early auxin gene expression, or that have altered seed development.
In one aspect, the invention therefore features a method for increasing stress resistance or tolerance in a plant. The method, in general, includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby increasing the level of stress resistance or tolerance in the transgenic plant. In preferred embodiments, the expression of the DNA encoding the kinase domain activates the expression of a stress-inducible gene (e.g., a gene encoding a glutathione S-transferase, an asparagine synthetase, or a heat shock protein). In particular applications, the method is especially useful for providing to a plant resistance or tolerance to an environmental stress. Exemplary environmental stresses include, without limitation, those which occur upon exposure of the transgenic plant to limited or inadequate water availability (e.g., drought conditions), excess salt or osmotic conditions, excess temperature conditions (e.g., heat, cold, or frost), excess light, a pathogen, a chemical (e.g. a metal, herbicides, and pollutants), an oxidative stress, UV light, and wounding. In preferred embodiments, the plant is protected against multiple stress conditions.
In another aspect, the invention features a method for reducing the action of an auxin in a plant. The method includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby reducing the action of the auxin in the transgenic plant. In preferred embodiments, the expression of the DNA encoding the kinase domain represses the expression of an early-auxin gene (e.g., those which are under the control of a promoter which is substantially identical to the GH3 promoter or a promoter which includes the ER7 element).
In still another aspect, the invention features a method for altering seed development. In particular, the method includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby altering the development of a seed in the transgenic plant. In preferred embodiments, the expression of the DNA encoding the kinase domain enriches endosperm development, enriches embryo development, or attenuates seed development. In yet other preferred embodiments, the attenuation of the seed development results in a seedless plant (e.g., a seedless fruit or vegetable).
In yet another aspect, the invention features a method for increasing the yield or productivity of a transgenic plant. The method generally includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby increasing the yield of the transgenic plant.
In related aspects of the invention, the invention features a plant (or plant cell, plant tissue, plant organ, or plant component) including a recombinant transgene capable of expressing a kinase domain of a MAPKKK, wherein the transgene is expressed in the transgenic plant under the control of a promoter that is functional in a plant cell. In preferred embodiments, the transgene includes a kinase domain which is obtained from a plant. In yet other preferred embodiments, the invention features a kinase domain which is obtained from a fungus (e.g., a yeast) or an animal (e.g., a mammal). In still other preferred embodiments, the transgene consists essentially of the kinase domain.
In related aspects, the invention features seeds and cells from a plant which include a recombinant transgene capable of expressing a kinase domain of a MAPKKK.
In still other related aspects, the invention features a vector (e.g., an expression vector) including a promoter functional in plant cells operably linked to a gene encoding a MAPKKK polypeptide and a cell (e.g., a plant cell or a prokaryotic cell such as Agrobacterium) that includes the vector. In preferred embodiments, the gene encodes a polypeptide that consists essentially of a kinase domain of a MAPKKK (e.g., a kinase domain from a plant MAPKKK such as NPK1 or an ANP) or a genetically engineered chimeric polypeptide that includes such a kinase domain.
In general, the kinase domain used in the methods or plants (e.g., transgenic plants or plants that are bred using a transgenic plant) of the invention is generally expressed by itself, as a MAPKKK polypeptide or kinase domain-containing fragment thereof, or as part of a genetically engineered chimeric polypeptide. Useful kinase domains include those that are capable of activating a gene involved in a stress response, repressing early auxin gene expression, or altering seed development. Exemplary kinase domains include, without limitation, those that are substantially identical to the kinase domains of NPK1 or an ANP (e.g., ANP1, ANP2, or ANP3) or AtMEKK1. Preferably, the methods and plants of the invention specifically utilize the kinase domain of NPK1 or ANP1. In other preferred embodiments, a full-length MAPKKK polypeptide or a kinase domain-containing fragment thereof that is substantially identical to any one of NPK1, ANP1, ANP2, or ANP3 is utilized.
The DNA encoding the kinase domain is, in general, constitutively expressed. However, if desired, the kinase domain is inducibly expressed, or such a domain is expressed in a cell-specific, tissue-specific, or organ-specific manner. Moreover, the kinase domain can also be expressed under cycling conditions (e.g., cell cycle or circadian conditions).
Exemplary plants which are useful in the methods of the invention, as well as for generating the transgenic plants (or plant cells, plant components, plant tissues, or plant organs) of the invention, include dicots and monocots, such as sugar cane, wheat, rice, maize, sugar beet, barley, manioc, crucifer, mustard, potato, soybean, sorghum, cassava, banana, grape, oats, tomato, millet, coconut, orange, rye, cabbage, apple, eggplant, watermelon, canola, cotton, carrot, garlic, onion, pepper, strawberry, yam, papaya, peanut, onion, legume, bean, pea, mango, and sunflower.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 40%, preferably 50%, more preferably 80%, and most preferably 90%, or even 95% sequence identity to a reference sequence (for example, the amino acid sequences of the kinase domains or full-length MAPKKK polypeptides of NPK1, ANP1, ANP2, or ANP3 or to their respective nucleic acid sequences (FIGS. 11, 12, 13, 14, 15, and 16; SEQ ID NOS: 7-22). For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or greater. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides or greater.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, FastA, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9cobtained fromxe2x80x9d is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic DNA, or combination thereof).
By xe2x80x9crecombinantxe2x80x9d is meant a nucleic acid (e.g., DNA) that, is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene. The term therefore includes, for example, a gene or fragment thereof that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a nucleic acid which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a MAPKKK kinase domain (e.g., NPK1, ANP1, ANP2, or ANP3).
By xe2x80x9creporter genexe2x80x9d is meant a gene whose expression may be assayed; such genes include, without limitation, xcex2-glucuronidase (GUS), luciferase (LUC), chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), and xcex2-galactosidase.
By xe2x80x9ca promoter functional in a plant cellxe2x80x9d is meant any minimal sequence sufficient to direct transcription in a plant cell. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements or chemical inducers) or elements that are capable of cycling gene transcription; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene or engineered into a transgene construct.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9cplant cellxe2x80x9d is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein, includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a nucleic acid sequence (e.g., a recombinant DNA sequence) which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into the nuclear or plastidic genome.
By xe2x80x9cincreasing stress resistance or tolerancexe2x80x9d is meant mediating a level of endurance, adaptability, or durability to a stress (e.g., a man-made stress, such as pollution, or an environmental stress, such as drought, salinity, and oxidative and temperature stresses) in a transgenic plant which is greater than that exhibited by a control plant (for example, a non-transgenic plant). Preferably, the level of stress resistance or tolerance in a transgenic plant (or transformed plant cell, plant component, plant tissue, or plant organ) of the invention is at least 5%, 10%, or 20% (and preferably 30% or 40%) greater than the tolerance to a stress exhibited in a non-transgenic control plant (or control plant cell, plant component, plant tissue, or plant organ). In other preferred embodiments, the level of stress resistance or tolerance to a stress is 50% greater, 60% greater, and more preferably even 75% or 90% greater than a control plant, with up to 100% above the level of tolerance as compared to a control plant being most preferred. The level of stress resistance or tolerance is measured by conventional methods used to determine plant growth and response to stress. For example, the level of stress tolerance to salinity may be determined by comparing physical features and characteristics (for example, plant height and weight, leaf area, plant water relations, ability to flower, ability to generate seeds, and yield/productivity) of transgenic plants and non-transgenic control plants.
The invention provides a number of important advances and advantages for the protection of plants against environmental stress, such as drought, salt, oxidative damage, and temperature. In addition, the invention provides a means for blocking auxin-inducible gene expression and its concomitant responses affecting plant growth and development. Furthermore, the invention is useful for altering seed development (e.g., for the production of seedless fruits or vegetables), as well as for manipulating endosperm or embryo development. Furthermore, the methods of the invention are advantageous because a kinase domain of MAPKKK is relatively unstable which allows for convenient transgene manipulation, thereby avoiding undesirable side effects
Moreover, the invention facilitates an effective and economical means to improve agronomically important traits of plants for tolerating the effects of dehydration, salinity, cold, and heat. The invention provides for increased production efficiency, as well as for improvements in quality and yield of crop plants and ornamentals. Thus, the invention contributes to the production of high quality and high yield agricultural products; for example, fruits, ornamentals, vegetables, cereals, and field crops. Genetically-improved seeds and other plant products that are produced using plants expressing the genes and methods described herein also render farming possible in areas previously unsuitable for agricultural production. The invention further provides a means for mediating the expression of stress-related protective proteins (e.g., glutathione S-transferase, asparagine synthetase, or a heat shock protein) that enable a plant to tolerate the effects of environmental stress. For example, transgenic plants constitutively expressing a kinase domain of a MAPKKK are capable of turning on a plant""s stress signal transduction pathway by activating the expression of multiple stress-related proteins, which, in turn, enhances the plant""s tolerance to multiple stress conditions. Expression of these gene products therefore obviates the need to express individual stress-related genes as a means to promote plant defense mechanisms against adverse environmental conditions.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.